Activation of telomerase is a hallmark of cancer in the early stages of tumorigenesis and is associated with telomere elongation, genetic instability, and subsequent immortalization of cells. There are several strategies for overcoming activated telomerase that are potentially useful for therapeutic treatment, including targeting the telomerase holoenzyme, telomeric G-quadruplexes with small molecules, human telomerase reverse transcriptase (hTERT), and human telomerase RNA, and using immune therapy.
hTERT is a catalytic subunit of telomerase and a critical element for telomerase activity. Expression of hTERT is not usually activated in normal cells, although other components of telomerase are expressed. In addition, hTERT has various telomere-independent functions, including enhancement of cellular proliferation, DNA damage response through change in chromatin structure, and inhibition of apoptosis by upregulation of BCL2 expression. These functions are independent of each other.
Overexpression of hTERT for cell immortalization or telomerase activation occurs in several ways, including increased gene copy number and modulation at the transcription level. At the transcription level, the hTERT promoter does not have TATA or CAAT boxes but does have several transcription factor binding sites within 1 Kb of the transcription start site and is controlled by epigenetic changes, such as chromatin remodeling or methylation of the CpG islands in the promoter region. With this transcription machinery, 0.004 RNA molecules per cell in telomerase-negative cells are elevated to 0.2-6 RNA molecules per cell in telomerase-positive tumor-derived cells, showing a strong correlation between telomerase activity and hTERT transcription level.
The essential region for activation of transcription is at the core promoter region, −181 base pairs from the transcription start site. This region includes the E-box for MYC and other elements for transcription activation. An additional upstream region likely contains transcription-repressing elements, because the longer promoter region shows decreased promoter activity. This core promoter region becomes nuclease sensitive during cell proliferation. Because the hTERT core promoter is selectively activated in cancer cells, it is targeted for gene therapy by utilizing the promoter for expression of cytotoxic tumor-suppressing proteins.
The present inventors have previously shown, by various biochemical experiments including DMS footprinting experiments, that end-to-end stacked G-quadruplex structures are formed in the core promoter element from 12 G-tracts. One of these structures has a unique 3:26:1 loop configuration; the 26-base internal loop is a hairpin structure and responsible for the unique cooperative folding of this G-quadruplex, which is believed to be important in transcription silencing. Stabilization of this G-quadruplex structure using small molecules causes repression of hTERT promoter activity. Significantly the mouse TERT lacks these 12 G-tracts and has a 16-fold higher transcriptional activation level.
Several groups have recently demonstrated that many different kinds of tumors have somatic mutations within the hTERT promoter region at positions −124, −124/125, −138/139, and −146 from the ATG start site. A G-to-A mutation (G/A) in the antisense strand is proposed to generatean ETS/TCF element that would increase binding of the ETS transcription factor for activation of hTERT transcription. Significantly, these mutations are also localized in the G-quadruplex with the 3:26:1 loop configuration. While it has been demonstrated in a number of oncogene promoters that the G-quadruplex functions as a silencer element, it has also been shown recently that, in the case of BCL2, the i-motif can act as a transcription activator, validating both secondary DNA structures as transcriptional targets for modulation of gene expression. Therefore, it can be reasonably inferred that DNA structural changes to either a G-quadruplex or an i-motif as a result of these mutations would also affect the transcription activity of the mutated hTERT promoter as well as the binding of the ETS transcription factor to the duplex form. Accordingly, modulation of transcription activity of the mutated hTERT promoter can be used to treat cancer as well as other clinical conditions associated with a transcription-activating mutationin an hTERT core promoter region or hTERT overexpression due to genomic rearrangements such as translocation or amplification.
Therefore, there is a need for a compound that can modulate transcription activity of hTERT to treat cancer and other clinical conditions associated with transcription or overexpression of hTERT.